Stock No: 012810
Protocol 23928: Sanger sequencing Assay - Enpp1<asj>
Version 1.2

Notes

The genotyping protocol(s) presented here have been optimized for reagents and conditions used by The Jackson Laboratory (JAX). To genotype animals, JAX recommends researchers validate the assay independently upon receipt of animals into their facility. Reaction cycling temperature and times may require additional optimization based on the specific genotyping reagents used.

Expected Results

Sequence

ATGGGAAGGGAGAGAAAGAGAGAGAAATAAATAAAACTTTTTTTTTCATTGTTA
ATACTAAATTTGTAGCACTTGTGTTACTTTTGACTTGTTCTGAATTTTGATCTGGC
TGTTTGATCTGCATCCTGGGATAATTTACTTAATGCGATATGCCTAATAGCAGTGT
CTGTTGTTGTTTTGTTTTCTGTCTAGAAAACTGTGGAACATACACTAAAAACATG
AGGCCTATGTACCCTACCAAGACGTTTCCCAATCATTACAGCATCG(t/a)CACAGTA
AGTAGGCACTAGGCACACTCTGTGTTTGGACCAGGAAAATGTAGTTGAAGCAC
TGTTCCAGGGCATAATTCAACTTCAGATGAGCATGAGGTAATTTGTTTTGAAATT
CAAGCTATAATCTGCAATTGGTCTTTCCTTAGGGACTTTATCCGGAATCCCATGG
CATAATTGATAACAAGATGTATGATCCCAAAATGAATGCTTCTTTCTCGCTTAAAA
GTAAAGAGAAATTCAACCCTTTGTGGTACAAAGGACAGCCGGTAAGCTACTTCTT
GTGGCTACTAAAATAGTTTGTTATCCATACTTATCTTAAT

JAX Protocol

Protocol Primers

Primer 5' Label Sequence 5' → 3' 3' Label Primer Type Reaction Note
13013 TGA TCT GCA TCC TGG GAT AA Forward A
13014 TAA GGA AAG ACC AAT TGC AGA Reverse A

Reaction A

Component Final Concentration
ddH2O
Kapa 2G HS buffer 1.30 X
MgCl2 2.60 mM
dNTPS-kapa 0.26 mM
13013 0.50 uM
13014 0.50 uM
Glycerol 6.50 %
Dye 1.00 X
Kapa 2G HS taq polym 0.03 U/ul
DNA

Cycling

Step Temp °C Time Note
1 94.0 --
2 94.0 --
3 65.0 -- -0.5 C per cycle decrease
4 68.0 --
5 -- repeat steps 2-4 for 10 cycles (Touchdown)
6 94.0 --
7 60.0 --
8 72.0 --
9 -- repeat steps 6-8 for 28 cycles
10 72.0 --
11 10.0 -- hold
JAX uses a very high speed Taq (~1000 bp/sec), use cycling times recommended for your reagents.
JAX uses a 'touchdown' cycling protocol and therefore has not calculated the optimal annealing temperature for each set of primers.

Strains Using This Protocol

This is the only strain that uses this protocol.